freestyle thermelute

8/16/2019 Freestyle Thermelute

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Visit us at www.LCTech.de!

Aflatoxin & Ochratoxin Analysis

A Completely Automated Solution with the

Freestyle, ThermELUTE, and SMARTImmunoaffinity Cleanup Columns


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Background

• Pickering Labs primarily makes HPLC Post-column

instruments and reagents for a variety of post-columnapplications.

• LC Tech has been the European distributor for Pickering Labs

for about 20 years.

• Recently Pickering Labs has been distributing LC Tech

products in North America along with help from

Chromatographic Specialties in Canada.


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What LCTech does

supplies innovative products

and methods for thepreparation and analysis of

food, feed and environmental

samples.


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Introduction

The goal: totally automated system

for Aflatoxins (B1, B2, G1, G2) and/or Ochratoxin A

HPLC

From raw extract to chromatogram

=> in 15 minutes

=> without manual steps


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Introduction

The Components

UVETM

UV derivatiser (only for Aflatoxins)

ChromatogramFREESTYLETM with ThermELUTE

and SPE modules

Raw extract

quaternary or

isocratic pump

sampler

column

oven

detector

HPLC


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Instrumentation

Sample preparation Analysisand

FREESTYLE Basic

SPE module

ThermELUTE module

Racks and vials

AflaCLEAN and/or OtaCLEAN

SMART Immunoaffinity Cleanup

Columns

Any HPLC

Pump(s)

Autosampler

Column oven

Derivatizer (e. g. UVETM)

Fluorescence detector


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Sample preparation with large volume injection

FREESTYLE Basic Automated and unattended handling

of the immunoaffinity column:

- loading of a precise volume

- washing

- drying in case of AflaCLEAN SMART

- column transfer into the ThermELUTE module

ThermELUTE module heating = breaking the antigen-antibody bond

- elution of the analytes with water and

- large volume injection into HPLC sample loop


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Analysis Instrumentation

Any HPLC possible

tested with DIONEX Ultimate 3000

Pump Isocratic mode

Autosampler Only needed for standard injection

Column switching

valve

If Aflatoxins and Ochratoxin A should be analyzed

a switching valve is recommended

Column Oven For two columns (Afla & Ochra)Column(s) Standard guard and analytical columns

Derivatizer UV derivatizer, photochemical derivatization

Fluorescence detector Standard

Software Standard


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Dionex Ultimate 3000 details

Pump LPG 3400a

Sampler WPS 3000 TSL

Mobile Phase Methanol/Water/Acetonitrile, 30/60/15

Flow rate 1.2 mL/min

Column Oven 36 °C

Column LCTech HPLC column (LCTech P/N 10522)

Flourescence detector RF 2000, FLD

Fluorescence wavelength 365 nm excitation

460 nm emission

Derivatizer LCTech UVE

ThermELUTE – chromatograms


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The goal:

Completely automated system

for Aflatoxin and/or Ochratoxin A analysis

Step 1:

Replace 1 mL or 3 mL immunoaffinity columnswith SMART Immunoaffinity columns


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What is so smart about SMART columnns?

SMART columns - advantages

Original size:25 mm They are completely comparableto 1 mL or 3 mL immunoaffinity

columns

But,

-less t ime is needed-less sample is needed

-less solvent is needed

-Less waste

And-toxin concentration is equivalent

to 1 mL or 3 mL format

S M A R

T C o l u m n s


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They meet AOAC requirements


Loading capacity

100 ng B1

Loading capacity

100 ng Ochratoxin A

Recovery > 80 % Recovery > 80 %

S M A R

T C o l u m n s


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Reduced Cleanup time


Cleanup process down

to less than 9 minutes

Cleanup process down

to less than 13 minutes

Incredible time reduction

- less sample volume = less time for sample loading

- high flow rate

S M A R

T C o l u m n s


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They are applicable for all regulated matrices


All kinds of nuts:

Hazelnut, walnut, brazil nut,

peanut, etc.

pistachios, almonds, raisins, figs

All kinds of cereals, corn, rice

All common spices

etc.

All kinds of coffee

Red wine

Beer

Raisins

Liquoriceetc.

S M A R

T C o l u m n s


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The goal:

Completely automated system

for Aflatoxin and/or Ochratoxin A analysis

Step 1:

Replace 1 mL or 3 mL immunoaffinity columns

by SMART Immunoaffinity columns

Step 2:

High end Completely automated solution

with FREESTYLE and ThermELUTE module


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Completely automated solution

ThermELUTE

module

Rack for SMART columns

Any HPLC

SMART

columns

SPE Gripper


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Width:110 mm

ThermELUTE , standard version (p/n 13572)

Heating element

Injection valve


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Rack (p/n 13497)

for 60 SMART columns

Rack (p/n 13497) in tray (p/n 11915)

for SMART columns



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ThermELUTE with Standards

Injection valve

Injection port

Extraction and direct

injection system

Cooled rack for standards

SMART Column

Gripper


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ThermELUTE module

Rack for up to 60 SMART columns


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SMART Column Gripper


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How the systemoperates:

SMART Column

gripper is taken upby SPE tool


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gripper takes up the

SMART column

Used columns

Unused columns


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The SMART column

is now tightlyconnected

to the system and

can be manipulated

by positive pressurecontrol


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After handling steps

(loading, washing,drying…) column is

transfered into heating

element

Heating element (hidden)


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Column is transfered

into heating element

Heating element (hidden)

The antigen-antibody

bond is broken by the

heat and the toxins are

eluted with water. No

organic solvent is

required.


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Original size:

25 mm

Normally you elute with organic

solvent; either methanol or acetonitrile.

This destroys the antigen-antibody

bond and elutes the toxins. The

disadvantage is that you cannot inject

high volumes of methanol onto the

Reverse Phase column. You usually

have to evaporate your cleaned sample

and re-suspend in a small volume prior

to injection onto the Reverse Phase

column.

When you are eluting with water you

are injecting all the toxin that is bound

to the SMART column (~700ul) directly

onto the Reverse Phase column.

S M A R

T C o l u m n s


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How the system

operates:

Column is now

in the heating element

The toxins are eluted into the

injection valve after thermalantigen-antibody bond

cleavage.

Sample was eluted as a

partial loop fil l with 400 µLinto 700 µL sample loop

Sample loop

Injection valve

Injection port


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Elution followed by whole sample large volume injection

Advantages

No loss of analytes. No storage between cleanup andanalysis

Large volume injection in water / all analytes are injected

straight from the column

- No evaporation

- No adsorption by the glass wall of storage vials

- No loss of recovery = highest sentivity

Less sample required to stay within FLD range.

- Less matrix on the immunoaffinity column

- Faster sample clean-up


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Elution followed by whole sample large

volume injection

Advantages

Suitable for very strict detection limits

for baby food

Maximum level according

to EU Regulation1881/2006 *

Detection limit with

FREESTYLE +ThermELUTE

Aflatoxin B1: 100 ppt Aflatoxin B1: 5 ppt

Ochratoxin A: 500 ppt Ochratoxin A: 30 ppt

* COMMISSION REGULATION (EC) No 1881/2006 of 19 December 2006

setting maximum levels for certain contaminants in foodstuf fs


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Elution followed by whole sample large volume injection

Advantages

Single-use AflaCLEAN and OtaCLEAN SMART Cleanupcolumns

- 100% initial performance of SMART columns for all samples

- No cross contamination

= Perfect analytical conditions


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Increased sample throughput by parallelization of

sample preparation and analysis


FREESTYLE sample preparation time: 15 min* / sample

1st sample

1st sample

Sample preparation

with FREESTYLETM

Analysis

with HPLC

2nd sample

2nd sample 3rd sample

HPLC analysis time: 15 min* / sample

4rth … etc

3rd … etc

* Ochratoxin A; Aflatoxin even faster

Automated

sample injection


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Throughput at i ts best

Completely automated solution - advantages

15 min / sample

4 samples per hour

96 samples per day

Monday Thuesday

Monday – Sunday

Day and night


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Loading capacity:

90 samples(1 SMART column /

sample

& samples presented

in 16 mL vials)

Quantity of samples

can be increased by

reduction of sample

volume and by choosing

smaller vials.

Completely automated solution - advantages


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Higher sensitivity compared to manual cleanup processing

Excellent chromatograms (less sample needed)


Ginger (10 ppb) withThermELUTE, 2.8 mL sample

Ginger (10 ppb), manualprocessing, 14 mL sample

Ginger

0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0

10,0

0,0

10,0

20,0

30,0

40,0

50,0

60,0

1 - 130206-FW- SOFTWARE 5.2 U SYSTEMWASC Emissi

ingwer halbe einwaag EmissimV

min

2

1

EM:460 nm

0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0

-50

100

200

300

400

ingwer blind 2,8ml halbe einwaage Emission

ingwer 10ppb 2,8ml halbe einwaage EmissionmV

min

21

EM:460 nm


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Nutmeg (10 ppb) withThermELUTE, 2.8 mL sample

Nutmeg (10 ppb), manualprocessing, 14 mL sample

Spices: Nutmeg

0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0

-20

50

00

50

00

muskatnuss halbe einwa Emissi

muskatnuss halbe einwaa EmissimV

min

21

EM:460 nm

0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0

-50

0

50

100

150

200

250

300

muskatnuss blind 2,8ml halbe einwaageEmission

muskatnuss 10ppb 2,8ml halbe einwaageEmissionmV

min

21

EM:460 nm


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Coriander (10 ppb) withThermELUTE, 2.8 mL sample

Coriander (10 ppb), manualprocessing, 14 mL sample

Spices: Coriander

0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0

-10

20

40

60

80

10

1 - 130206-FW- SOFTWARE 5.2 U SYSTEMWASC Emissi

koriander halbe einwaa EmissimV

min

21

EM:460 nm

0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0

-50

0

50

100

150

200

250

300

koriander blind 2,8ml halbe einwaageEmission

koriander 10ppb 2,8ml halbe einwaageEmissionmV

min

21

EM:460 nm


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Even low levels are detected. Not detected with manual

processing


Hazelnut, not spiked

Black:

ThermELUTE detects evenlowest level

Red:

Not detected with manual

processing

Nuts: Hazelnut

0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0

-50

0

50

100

150

200

250

300

Haselnuss 10ml blind Emission

2 - 130205-FW-THERMELUTE AFLACLEAN PISTAZIE_HASELNUSS_ROSINE_ERDNUSS #50mV

min

21

EM:460 nm


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Even low levels are detected. Not detected with manual

processing


Peanut, not spiked

Black:

ThermELUTE detects evenlowest level

Red:

Not detected with manual

processing

Nuts: Peanut

0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0

-50

0

50

100

150

200

250

300

Erdnuss 10ml blind Emission

Erdnuss blind manuell 3ml EmissionmV

min

21

EM:460 nm


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Less sample needed, thus faster processing


Corn, 10 ppb

Black:

ThermELUTE, 2.5 mLRed:

ThermELUTE, 1 mL

Orange:

Manual processing, 3 mL

Cereals: Corn

0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0

-100

125

250

375

500

700

Probe 4 10ppb 2,5ml Emission

Probe 5 10ppb 1ml Emission

10ppb Probe mais manuell 3ml Säule EmissionmV

min

321

EM:460 nm


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Hazelnut, 10 ppb

Black:


ThermELUTE, 1 mL

Orange:


Nuts: Hazelnut

0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0

-100

125

250

375

500

700

Haselnuss 2,5ml 10ppb EmissionHaselnuss 1ml 10ppb Emission

Haselnuss 10ppb manuell 3ml EmissionmV

min

321

EM:460 nm


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Raisins 10 ppb

Black:


ThermELUTE, 1 mL

Orange:


Raisins

0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0

-100

125

250

375

500

700

Rosinen 2,5ml 10ppb EmissionRosinen 1ml 10ppb Emission

Rosinen 10ppb manuell 3ml EmissionmV

min

321

EM:460 nm


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Pistachio, 10 ppb

Black:


ThermELUTE, 1 mL

Orange:


Nuts: Pistachio

0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0

-100

125

250

375

500

700

Pistazien 2,5ml 10ppb EmissionPistazien 1ml 10ppb Emission

Pistazien 10ppb manuell 3ml EmissionmV

min

321

EM:460 nm


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Peanut, 10 ppb

Black:


ThermELUTE, 1 mL

Orange:


Nuts: Peanut

0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0

-100

200

400

600

800

Erdnuss 2,5ml 10ppb EmissionERdnuss 1ml 10ppb Emission

Erdnuss 10ppb manuell 3ml EmissionmV

min

321

EM:460 nm


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Chromatogram of Peanuts Spiked with 10ppb total aflatoxin(G2/G1/B2/B1) (1ng G2; 4ng G1; 1ng

B2; 4 ng B1) extracted according to

LCTech extraction and clean-up

procedure, 10 mL processed using

AflaCLEAN SMART and

ThermELUTE.

Chromatogram represents 0.28 gram

matrix equivalents.

Clean-up time consists of

10 mL sample loading (3mL/min)

2 mL washing (3mL/min)

6 min ThermELUTE treatmentReady to inject within 10 min

Methods - Aflatoxin

Nuts: Peanut

M th d Afl t i


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Chromatogram of Peanuts Spiked with 10ppb total aflatoxin(G2/G1/B2/B1) (1ng G2; 4ng G1;

1ng B2; 4 ng B1) extracted according

to LCTech extraction and clean-up

procedure, 2.5 mL processed using

AflaCLEAN SMART and

ThermELUTE.


matrix equivalents.


2.5 mL sample loading (3 mL/min)


6 min ThermELUTE treatmentReady to inject within 7 5 min

Methods - Aflatoxin

Nuts: Peanut

M th d Afl t i


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Spiked with 10ppb total aflatoxin

(G2/G1/B2/B1 (1ng G2; 4ng G1;



procedure, 10 mL processed using

AflaCLEAN SMART and

ThermELUTE.


matrix equivalents.


10 mL sample loading (3mL/min)


6 min ThermELUTE treatmentReady to inject within 10 min

Methods - Aflatoxin

Chromatogram of Hazelnuts

Nuts: Hazelnut

M th d Afl t i


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Spiked with 10ppb total aflatoxin

(G2/G1/B2/B1) (1ng G2; 4ng G1;



procedure, 2.5 mL processed using

AflaCLEAN SMART and

ThermELUTE.


matrix equivalents.


2.5 mL sample loading (3 mL/min)


6 min ThermELUTE treatmentReady to inject within 7 5 min

Methods - Aflatoxin

Chromatogram of Hazelnuts

Nuts: Hazelnut

Ochratoxin


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10 mL instant coffee sample after

extraction using methylene chlor idepartitioning, spiked at the regulated

level of 10 ppb.

The chromatogram represents

0.01 gram matrix equivalents.

By using PICKERING post-column

derivatization sensitivity could be

increased by a factor of approx. 4 and

chromatography (injection peak/matrix

interference could be dramatically

reduced).

Ochratoxin

Chromatogram of Instant Coffee

Coffee

Methods Ochratoxin


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10 mL roasted cof fee sample after

extraction using methylene chlor idepartitioning, spiked at the regulated

level of 5 ppb.

The chromatogram represents 0.05

gram matrix equivalents.

Methods - Ochratoxin

Chromatogram of Roasted Coffee

Coffee

Methods Ochratoxin


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10 mL White Wine sample extracted

according to LCTech OtaCLEANSMART extraction and clean-up

procedure loaded onto OtaCLEAN

SMART column, spiked at the

regulated level of 2 ppb.

The chromatogram represents 1 grammatrix equivalents.


Chromatogram of White Wine

Wine

Methods Ochratoxin


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10 mL Beer sample extracted

according to LCTech OtaCLEANSMART extraction and clean-up

procedure loaded onto OtaCLEAN

SMART column, spiked with 1 ppb

The chromatogram represents 1.42

gram matrix equivalents.


Chromatogram of Beer

Beer

Completely automated solution - further features


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SPE Module

EVAporationModule

GPC Module

ThermELUTE

Module

Completely automated solution - further features


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Visit us at www.LCTech.de!

www.freestyle-robotic.comwww.chromspec.com

[email protected]

[email protected]

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